Abstract
Background: The incidence of Type 2 diabetes mellitus (T2DM) combined
with non-alcoholic fatty liver disease (NAFLD) has risen over the
years. This comorbid condition significantly increases the probability
of cirrhosis, liver cancer, and mortality compared to the disease
alone. The multi-targeted, holistic treatment efficacy of traditional
Chinese medicine (TCM) plays a vital role in the treatment of T2DM and
NAFLD. Jiedu Tongluo Tiaogan Formula (JTTF), based on TCM theory, is
widely used in clinical treatment, and its effectiveness in lowering
glucose, regulating lipids, improving insulin resistance, and its
pathways of action have been demonstrated in previous studies. However,
the mechanism of this formula has not been investigated from a
metabolomics perspective. Moreover, high-quality clinical studies on
T2DM combined with NAFLD are lacking. Therefore, we aim to conduct a
clinical trial to investigate the clinical efficacy, safety, and
possible pathways of JTTF in the treatment of T2DM combined with NAFLD
using metabolomics techniques.
Methods: A total of 98 participants will be recruited to this clinical
trial and randomly assigned to either a treatment group (JTTF +
conventional basic treatment) or control group (conventional basic
treatment) in a 1:1 ratio. Both groups will have received the same
lifestyle interventions in the preceding 12 weeks. The primary outcome
will be change in visceral fat area and total score on the TCM
syndromes efficacy score scale. The secondary outcome will include
changes in ultrasound steatosis grade, fibrosis 4 score (FIB-4),
metabolic parameters, anthropometric parameters, visceral fat area. In
addition, serum and urine samples collected at baseline and at the end
of 12 weeks of treatment will be sequentially tested for untargeted and
targeted metabolomics.
Discussion: This study will evaluate the efficacy and safety of JTTF,
as well as investigate the differential metabolites and possible
mechanisms of JTTF treatment in T2DM combined with NAFLD. We
hypothesize that patients will benefit from JTTF, which may provide
strong evidence for the clinical use of JTTF in the treatment of T2DM
and NAFLD, leading to the possibility of further mechanistic
exploration.
Clinical Trial Registration: This clinical trial has been registered in
China Clinical Trial Registry (ChiCTR 2100051174).
Keywords: jiedu tongluo tiaogan formula, type 2 diabetes, non-alcoholic
fatty liver disease, randomized controlled trial, metabolomics,
traditional Chinese medicine
1 Introduction
The global burden of diabetes continues to rise annually among the
general population with a predicted increase from 10.5% (536.6 million
people) in 2021 to 12.2% (783.2 million) by the year 2045 in people
aged 20–79 years ([48]Sun et al., 2022). According to the
epidemiological survey conducted by Li et al. ([49]Li et al., 2020),
diabetes is prevalent in 11.2% of the Chinese population. Diabetes and
related complications significantly affect quality of life, mortality
and morbidity, and contributes to greater economic burden on
individuals and society at large ([50]American Diabetes Association,
2018). Non-alcoholic fatty liver disease (NAFLD) is one of the most
common chronic liver diseases worldwide and includes various
pathological states such as simple steatosis and non-alcoholic
steatohepatitis (NASH). The disease is initially accompanied by fat
accumulation and may gradually progress to other diseases such as liver
fibrosis, cirrhosis, or hepatocellular carcinomas ([51]Chalasani et
al., 2018). In the absence of any intervention for NAFLD, human health
is endangered and the risk of death increases by at least 71% ([52]Tilg
and Targher, 2021). Studies have shown that the prevalence of NAFLD in
patients with T2DM is greater than 55% ([53]Younossi et al., 2019),
which is 5–9 times higher than the incidence in the general population.
Conversely, individuals with NAFLD have a significantly higher risk of
developing diabetes than those without NAFLD, which is more than twice
as high as that of healthy individuals ([54]Mantovani et al., 2018).
More than 60% of patients were found to have abnormal glucose
metabolism, leading to a 47.3% probability of diabetes ([55]Buzzetti et
al., 2016); thus NAFLD may be an early predictor of T2DM ([56]Lee et
al., 2019).
The pathogenesis of T2DM and NAFLD is complex and not yet fully
understood. However, it is widely accepted that the two diseases share
a common pathogenesis, insulin resistance (IR), both as a cause and a
consequence of T2DM combined with NAFLD ([57]Park et al., 2013).
Clinical trials developing drugs to treat NAFLD have also focused on
the discovery of insulin sensitizers ([58]Ibrahim et al., 2013). Many
of the same changes have been shown to occur in the development of both
diseases, including abnormal glucose and lipid metabolism, genetic
susceptibility, environmental influences, and lifestyle changes
([59]Saponaro et al., 2015). NAFLD is usually treated as a benign
disease and neglected for management and treatment. However, when it
occurs in combination with diabetes, it exacerbates imbalances in
glucolipid metabolism, and abnormalities in glucose and lipid
metabolism interact to affect each other. Moreover, diabetes increases
the risk of cirrhosis and mortality in patients with NAFLD, while NAFLD
increases the incidence of cardiovascular disease and all-cause
mortality in patients with T2DM ([60]Radaelli et al., 2018). In the
presence of abnormalities in the relevant metabolic and anthropometric
indicators, clinical screening for disease should be performed promptly
to exclude them. The occurrence of this comorbidity is usually
accompanied by abnormalities in glucolipid metabolism-related
indicators such as glucose, lipids, and insulin levels ([61]Ziolkowska
et al., 2021). Also, some anthropometric indicators are closely
associated with the occurrence and development of comorbidity,
represented by WHtR ([62]Swainson et al., 2017). Therefore, it makes
sense to adopt a holistic approach to the treatment of these metabolic
diseases with highly related pathogenesis and pathological
manifestations.
Changes in molecular products in the human body can precede the
appearance of clinical symptoms ([63]Wu et al., 2019); hence,
metabolomics has been gradually applied in recent years to explore
disease diagnosis, pathogenesis, and risk assessment ([64]Roberts et
al., 2014; [65]Newgard, 2017, [66]2017; [67]Wishart, 2019; [68]Schmidt
et al., 2021). This technique may also identify biomarkers for the
early diagnosis of a disease ([69]Esperanza et al., 2020; [70]Cui et
al., 2021) or explore metabolic differentials in the action of Chinese
and Western drugs ([71]Tao et al., 2019; [72]Wu et al., 2019; [73]Xiao
et al., 2020; [74]Xu et al., 2020; [75]Luo et al., 2021; [76]You et
al., 2021). Further, it has applications for assessing the variability
of molecular metabolic products in patients with different traditional
Chinese medicine (TCM) syndromes of the same disease ([77]Guo et al.,
2021; [78]Shan et al., 2021). Other studies have applied metabolomic
techniques to the diagnosis and mechanistic exploration of diabetes and
its complications ([79]Jimenez-Luna et al., 2020; [80]Wang S. et al.,
2021; [81]Zhang F. et al., 2021; [82]Tan et al., 2021), and to NAFLD
([83]Masoodi et al., 2021; [84]Piras et al., 2021). Therefore, it is
feasible and relevant to study the treatment of this comorbid disease
at a molecular level.
The current Western medical treatment of T2DM combined with NAFLD is
mainly aimed at reducing fat accumulation in the liver and delaying the
onset of inflammation and fibrosis ([85]Ks et al., 2019), and relies on
lifestyle management, the application of glucose- and lipid-lowering
drugs, and the implementation of bariatric surgery. Glucose management
is particularly important for inhibiting the development of liver
fibrosis ([86]Nakahara et al., 2014). Lifestyle modification is a basic
form of treatment for patients with T2DM and NAFLD. Reducing body mass
index (BMI) can effectively improve liver function and insulin
resistance levels ([87]Al-Jiffri et al., 2013). However,
non-pharmacological therapies often show low compliance in the patient
population and are difficult to adhere to. Although there are currently
no Food and Drug Administration (FDA)-approved drugs for NAFLD or NAFLD
combined with type 2 diabetes ([88]Ferguson and Finck, 2021), many
emerging drug therapies such as targeted therapies are in development
([89]Negi et al., 2022). The continuous clinical exploration of drugs
for the treatment of NAFLD in combination with T2DM shows the high
incidence of this comorbidity and the necessity of treatment. There are
many glucose-lowering drugs also used in the treatment of NAFLD in the
clinic, such as glucagon-like peptide-1 (GLP-1), Thiazolidinediones,
Metformin, which have good effect on fat reduction and glucose
reduction ([90]Negi et al., 2022). However, there are still some
limitations, for example, metformin has a weak effect on hepatic fat;
thiazolidinediones may show weight gain and female fractures despite
their insulin-sensitive effects; GLP-1 has a significant weight loss
effect but is often accompanied by gastrointestinal side effects. In
summary, additional therapeutic strategies are urgently required to
treat this complicated disease.
TCM treats diseases from a holistic viewpoint, harmonizing the organs
of the whole body in a comprehensive manner. This medicine system has
multi-component, multi-target, multi-pathway, prospective, and stable
characteristics, and is especially effective in treating complex and
variable chronic diseases such as T2DM ([91]Wang X. et al., 2021).
Chinese medicine can improve IR and glucolipid metabolic function,
among other effects, by regulating the inflammatory response and
oxidative stress capacity, and improving the structure of the
intestinal flora, thereby treating T2DM combined with NAFLD. It not
only improves the clinical index of patients but also alleviates
clinical symptoms for the purpose of prevention and treatment. For
example, the Chinese botanical drugs such as Shengmaiyin and Ganmai
Dazao Tang ([92]Fan et al., 2018; [93]Li et al., 2019), and other
appropriate Chinese medicine techniques such as acupuncture and
acupuncture point burial are widely used in clinical practice to assist
in lowering sugar and reducing fat ([94]Li et al., 2021; [95]Wang et
al., 2022).
Jiedu Tongluo Tiaogan Formula (JTTF) is a clinically effective formula
based on TCM theory, consisting of the dried rhizome of Coptis
chinensis Franch [Ranunculaceae; Coptidis rhizome], the dried rhizome
of Rheum palmatum L [Polygonaceae; Rhei radix et rhizoma], the dried
root of Astragalus mongholicus Bunge [Fabaceae; Astmgali radix], the
dried rhizome of Salvia miltiorrhiza Bunge [Lamiaceae; Salviae
miltiorrhizae radix et rhizoma], the dried root of Bupleurum chinense
DC [Apiaceae; Radix bupleuri], at the weight ratio of 15:9:15:15:10.
This formula is involved in detoxifying and regulating the draining
function of the liver to eliminate the “toxic evil” in the body for
disease prophylaxis and treatment. The effectiveness of this formula in
lowering blood glucose, regulating blood lipids, promoting insulin
secretion, and improving IR has been verified in many basic and
clinical trials.
The main components of JTTF have been analyzed by High Performance
Liquid Chromatography (HPLC) in our previous study ([96]Zhang Q. et
al., 2021), which can be seen in [97]Supplementary Material S1. On the
basis of reference standard data, chromatographic elution behavior,
chemical composition data, mass fragmentation pattern and HPLC results,
the chemical components of JTTF were identified as chlorogenic acid,
anthocyanin-7-glucoside, tannic acid B, aloe rhodopsin and haragoside.
By means of the validation of network pharmacology and in vitro
experiments, it proves that JTTF plays an important role in promoting
insulin secretion and regulating glucolipid metabolism by acting on the
PI3K-Akt pathway, an important pathway for insulin signaling and
glucose regulation ([98]Zhang Q. et al., 2021). Related research has
demonstrated that JTTF can inhibit the expression of inflammatory
factors associated with endoplasmic reticulum stress, thereby
alleviating this condition ([99]Jiang et al., 2018), improving IR, and
increasing insulin sensitivity ([100]Liu et al., 2021). At the same
time, it can activate the IRE1α/JNK pathway, which is closely related
to IR and pancreatic β-cell apoptosis to reduce the apoptosis of
pancreatic islet cells and prevent and treat T2DM ([101]Piao et al.,
2018). Studies showed that blood glucose and lipid levels, as well as
IR levels of diabetic ZDF rats were significantly improved by high-dose
JTTF ([102]Liu et al., 2021). Related clinical studies have shown that
JTTF may reduce glycated hemoglobin, blood glucose, and inflammatory
factor levels, regulate lipid and insulin resistance levels in T2DM
patients ([103]Jin et al., 2020), and have significant efficacy in
reducing liver function, BMI, waist circumference, and TCM symptom
scores in T2DM combined with NAFLD patients ([104]Gong, 2017).
Herein we investigate the clinical efficacy and safety of JTTF for the
treatment of T2DM combined with NAFLD. It is necessary to design and
conduct this trial study as the mechanism of action of this formula has
not been explored at the molecular level.
2 Methods and analysis
2.1 Study objectives
2.1.1 Primary objective
The primary objective of this clinical trial is to assess the efficacy
and safety of JTTF for the treatment of T2DM combined with NAFLD. We
hypothesized that JTTF combined with conventional Western medicine
would be more effective than the single use of conventional Western
medicine alone in reducing fat accumulation, inhibiting liver fibrosis,
regulating blood lipids, lowering blood glucose levels, and alleviating
the clinical symptoms of patients with T2DM and NAFLD.
2.1.2 Secondary objective
The secondary objective of this study was to identify differential
metabolites in the serum and urine of patients before and after JTTF
treatment to determine possible relevant metabolic pathways and routes
of action in T2DM combined with NAFLD. Furthermore, we explored the
regulatory effects and mechanisms of JTTF action on various aspects
including lipid and glucose regulation at the metabolic level.
2.2 Study design
This study is designed as a 12-weeks, single-center, double-armed,
parallel, randomized, controlled clinical trial. A total of 96 eligible
participants will be recruited from Shenzhen Hospital of Guangzhou
University of Chinese Medicine (Futian) and randomized in a 1:1 ratio
into two groups to receive basic treatment. As a supplement to the
clinical study, participants’ fasting serum and urine samples will be
subjected to untargeted and targeted metabolomic assays. Liquid
chromatography–mass spectrometry (LC-MS) will be used to compare
metabolites before and after treatment in the experimental and control
groups. Basic analysis of metabolites, multivariate statistical
analysis, pathway enrichment analysis, and topological analysis of the
screened differential metabolites will be performed.
All patients should have voluntarily participated and signed an
informed consent form with good compliance. Informed consent will be
obtained from all participants prior to enrollment. A flowchart and
schedule of the clinical trial is shown in [105]Figure 1 and [106]Table
1, respectively. The protocol was designed according to the Standard
Protocol Items: Recommendations for Interventional Trials (SPIRIT)
guidelines ([107]Chan et al., 2013) and the Declaration of Helsinki.
The SPIRIT checklist is shown in [108]Supplementary Material S2.
FIGURE 1.
[109]FIGURE 1
[110]Open in a new tab
Flow chart of the clinical trial.
TABLE 1.
Schedule of the clinical trial.
Project time Screening/baseline
Treatment
-14–0 days 0±2 days Visit 1 4weeks± 2days Visit 2 6weeks± 4days Visit 3
8weeks± 6days Visit 4 12weeks± 7days
Eligibility screening
Inclusion/exclusion criteria ✓
Informed consent form ✓
Allocation ✓
General information ✓
Family history ✓
Medical history ✓
Drinking history ✓
Physical examination^a ✓ ✓ ✓ ✓ ✓ ✓
Vital signs^b ✓ ✓ ✓ ✓ ✓ ✓
Ultrasound of liver ✓ ✓
bioelectrical impedance analysis
visceral fat area ✓ ✓
subcutaneous fat area ✓ ✓
Electrocardiography ✓ ✓ ✓
Laboratory examination
Routine blood test ✓ ✓ ✓
Routine urine test ✓ ✓ ✓
Fasting blood sugar ✓ ✓ ✓
2-h plasma glucose ✓ ✓
Glycated hemoglobin ✓ ✓
Fasting insulin ✓ ✓
Blood lipids ✓ ✓ ✓
Liver function ✓ ✓ ✓
Renal function ✓ ✓ ✓
Traditional Chinese medicine symptoms ✓ ✓ ✓ ✓ ✓
Fibrosis 4 Score ✓ ✓
Homeostasis model assessment-insulin resistance ✓ ✓
homeostatic model assessment for β-cell ✓ ✓
Metabolomics test^c
Serum sample ✓ ✓
Urine sample ✓ ✓
Adverse event ✓ ✓ ✓ ✓
Prescription ✓ ✓ ✓ ✓ ✓
Combined drugs ✓ ✓ ✓ ✓ ✓
Summary of the analysis ✓
[111]Open in a new tab
2.3 Ethics and dissemination
This clinical trial has been approved by the Ethics Committee of
Shenzhen Hospital of Guangzhou University of Chinese Medicine (Futian)
(Project number: GZYLL(KY)-2021–025).
Prior to signing the informed consent form, the investigator will
provide the patient with a detailed description of the process,
potential risks and benefits of participating in the study.
Participants will sign an informed consent form and allow the study
results to be published in a peer-reviewed journal. All participants’
personal data will be kept confidential during and after the trial.
Only authorized researchers have access to all data on participants.
2.4 Inclusion criteria
* 1) All participants must meet the individual diagnostic criteria
for T2DM and NAFD. The diagnostic criteria for T2DM ([112]American
Diabetes Association Professional Practice Committee, 2022)
includes:
+ • Fasting plasma glucose (FPG) ≥126 mg/dl (7.0 mmol/L) or 2-h
plasma glucose (2-h PG) ≥200 mg/dl (11.1 mmol/L) during an
oral glucose tolerance test or HbA1c ≥ 6.5% (48 mmol/mol).
+ • Typical symptoms of hyperglycemia (excessive thirst,
polyuria, weight loss, hunger, pruritus, or coma) or
hyperglycemic crisis plus a random plasma glucose (PG)
≥200 mg/dl (11.1 mmol/L). Patients without typical symptoms of
diabetes need to be re-examined for confirmation. Fasting
state: no intake of calories for at least 8 h; random plasma
glucose: blood glucose at any time of day, regardless of the
last meal time (cannot be used to diagnose abnormal fasting
blood glucose or abnormal glucose tolerance). The diagnostic
criteria for NAFLD ([113]European Association for the Study of
the Liver (EASL) et al., 2016) ([114]Chalasani et al., 2018)
requires:
+ • Evidence of excess liver fat accumulation (hepatic
steatosis) on ultrasound imaging.
+ • Exclusion of excessive daily alcohol consumption ≥30 g for
men and >20 g for women ([115]Ratziu et al., 2010).
+ • Acute or chronic viral hepatitis (e.g., hepatitis C),
autoimmune hepatitis, hemochromatosis, Wilson’s disease,
alcoholic liver disease, drug-induced liver disease, liver
cirrhosis, inborn errors of metabolism (e.g., cholesterol
ester storage disease), or other causes of chronic liver
disease.
* 2) All participants must meet the diagnostic criteria for heat
stagnation syndrome in the liver and stomach in accordance with the
Guidelines for Clinical Research of Chinese Medicine (Zheng, 2002)
outlined below.
+ • Any two main symptoms including dry mouth, bitter and sticky
mouth, heaviness in the head and body, and/or obesity
+ • Any two secondary symptoms including fullness and distention
of the chest or abdomen, upset or irritable demeanor, rapid
digestion of food and polyorexia, deep-colored or turbid
urine, and/or unpleasant or constipated stools.
+ • Tongue condition: red tongue, yellow fur.
* • Pulse condition: stringy and rapid pulse or slippery and rapid
pulse.
* 3) Participants should be between the ages of 20 and 75 years.
* 4) The experimental group should not have taken traditional Chinese
medicine for diabetes and NAFD orally for at least 2 weeks before
enrollment.
2.5 Exclusion criteria
* 1) Patients with liver or kidney dysfunction: aspartate
transaminase [AST] or alanine transaminase [ALT] >2.5 times the
normal upper limit or creatinine >115 μmol/L.
* 2) Patients with chronic diseases, such as hypertension,
cardiovascular, cerebrovascular, autoimmune diseases, chronic
kidney diseases and psychiatric diseases who cannot follow research
procedures.
* 3) Patients who are using insulin.
* 4) Allergic constitution or a history of drug allergies.
* 5) Pregnant and lactating women.
* 6) Patients who participated in other clinical studies within the
preceding three months.
2.6 Drop-out criteria
* 1) Participants who would develop severe, acute conditions (e.g.,
diabetic ketoacidosis) or special physiological changes (e.g.,
state of pregnancy) that are not appropriate for this trial.
* 2) Participants who would show low compliance, such as failing to
take their medication regularly or at a rate of less than 80%
during the trial.
* 3) Participants who would have taken drugs prohibited by the study
protocol.
* 4) Participants who would voluntarily withdraw from the trial at
any time according to the informed consent form.
2.7 Intervention
The control group will receive conventional basic treatment, including
health education and oral medications to stabilize blood sugar, blood
pressure, and lipid levels. Meanwhile, the experimental group will be
given JTTF on the top of the control group. In this study, Chinese
herbal medicine concentrated granules (CCMG) will be used to replicate
the traditional method of preparing herbal tonics by using herbs as raw
materials and modern extraction and concentration techniques. All
herbal pellets will be manufactured and supplied by China Resources
Sanjiu Pharmaceutical Co. and also tested by their quality management
department for compliance with the grade standards of the Chinese
Pharmacopoeia (2020 edition). Voucher specimens of all drugs are
deposited at the Bureau of Traditional Chinese Medicine, Shenzhen
Hospital, Guangzhou University of Chinese Medicine (Futian) (Shenzhen,
China). The decoction and packing of botanical drugs are uniformly
operated by Kangmei Pharmaceutical Co., Ltd. Smart Pharmacy. CCMG is
based on the “Research Guidelines for Single Flavor Chinese Medicine
Concentrated Granules” and the “Pharmacopoeia of the People’s Republic
of China” (2010 edition). Extracted in a fully enclosed pipeline
production line in GMP workshop. All herbs are mixed in the proportions
described above, steeped in 10 volumes of pure water for 0.5 h, heated
to boiling, decocted for 1 h. The water extract is filtered and the
residue is then decocted with 8 volumes of pure water for 40 min. The
two liquid extracts are combined, settled and filtered, concentrated to
a thick paste, and processed into concentrated granules by spray
drying. The process parameters are screened by orthogonal design method
according to the composition characteristics of different species. A
certain amount of soluble dextrin is added as an auxiliary material to
assist the molding. The dry extract ratios of the five raw herbs were
1:6 for Coptis chinensis, 1:2.4 for Rheum palmatum, 1:10 for Astragalus
mongholicus, 1:6.7 for Salvia miltiorrhiza and 1:6 for Bupleurum
chinense. The final JTTF is made by combining the individual herbal
pellets in the leaching ratio, which is equivalent to 64 g of raw
herbs. It is packaged with aluminum-plastic-aluminum composite film
(BOPP/AL/CPE) packaging material sealed on four sides. Dissolve the
concentrated granules in 200 ml of boiling water and take warm. Take
twice daily, half an hour after breakfast and dinner. The observation
period will be administered for three months.
Both groups will receive the same health education information on
diabetes and NAFLD, assistance, lifestyle interventions, and
management. It includes developing a diet of low sugar, low fat, and
less refined carbohydrates, controlling the intake of total calories in
the diet, helping participants to choose appropriate exercise methods,
and encouraging patients to remain relaxed and actively cooperate with
treatment. Face-to-face nutritional education will be provided at each
follow-up visit.
2.8 Outcome assessment
2.8.1 Demographic information
All demographic information will be collected at enrollment, including
sex, age, course of disease, family history, previous medical history,
drug use, smoking, and drinking history.
2.8.2 Primary outcomes
The primary outcome is change in visceral fat area (VFA) and TCM
syndromes efficacy score scale. Changes in VFA will be compared between
the two groups before and after treatment. The indicator will be tested
at baseline and week 12. The TCM symptom scoring standard is presented
in [116]Supplementary Material S3.
2.8.3 Secondary outcomes
* 1) Changes in hepatic steatosis factors by ultrasound grading of
steatosis.
* 2) Changes in metabolic parameters: glycated hemoglobin (HbA1c)
levels, fasting plasma glucose (FPG), 2-h plasma glucose (2h-PG),
fasting insulin, blood lipids including total cholesterol (TC),
triglycerides (TG), high-density lipoprotein-cholesterol (c-HDL),
low-density lipoprotein (c-LDL), apolipoprotein A (ApoA), and
apolipoprotein B (ApoB).
* 3) Changes in anthropometric parameters: Waist-to-height ratio
(WHtR), Body Mass Index (BMI), waist circumference (WC), hip
circumference (HC).
* 4) Changes in hepatic fibrosis factors and hepatic function: The
changes in Fibrosis 4 Score (FIB-4,
[117]http://gihep.com/calculators/hepatology/fibrosis-4-score/) can
be calculated by the formula:
[MATH: Age (years)x AST /PLT (×109/L)xALT
:MATH]
The units of measurement for ALT and AST are aligned with those for age
and PLT.
Definitions for the following abbreviations: AST: Aspartate
aminotransferase; PLT: platelet; ALT: Alanine aminotransferase.
* 5) Changes in visceral fat area.
All measurements will be performed at baseline and at week 12.
Additional FPG and blood lipid test will be performed at week 6.
2.8.4 Monitoring outcomes
Homeostasis model assessment for insulin resistance (HOMA-IR) and
homeostatic model assessment for β-cell function (HOMA-β) will be
performed at baseline and at week 12.
2.8.5 Biological specimen analysis outcomes
Untargeted and targeted metabolomics tests will be performed
sequentially on fasting blood and urine samples collected at enrollment
and at the end of the 3-month treatment period. Biological samples will
be centrifuged and stored in a -80°C refrigerator. Assays will be
performed using liquid chromatography-mass spectrometry (LC-MS)
techniques for microscopic high-throughput measurements. Through basic
analysis, multivariate statistical analysis, and difference analysis,
the differential metabolites in serum and urine, before and after
treatment, will be compared between the two groups. Further targeted
metabolomic analyses will be performed to categorize differential
metabolites screened by untargeted metabolomics, as well as explore
their possible metabolic pathways. The exploratory results will be
combined with clinical efficacy evaluation to determine the mechanism
of action of JTTF in the treatment of T2DM complicated with NAFLD from
the perspective of metabolomics and small-molecule compounds.
2.8.6 Safety outcomes and adverse events
Safety assessments including vital signs, routine blood and urine
tests, 12-lead electrocardiogram, and hepatic function (AST/ALT/GGT)
and renal function tests will be conducted. General vital signs (body
temperature, heart rate, respiration rate, and blood pressure) will be
assessed at baseline and at weeks 4, 6, 8, and 12. Routine blood and
urine tests will be performed at baseline and weeks 6 and 12.
An adverse event (AE) is defined as any adverse medical event that
occurs between the time the participant signs the informed consent and
the last follow-up visit, whether or not it is causally related to the
study drug. Adverse events, such as subjective patient discomfort and
abnormalities in laboratory tests will be carefully analyzed and
investigated during each follow-up visit. Overall, JTTF is relatively
safe and may be associated with mild diarrhea. If this occurs, consider
taking half the dose for 2 days. Absence of remission or exacerbation
may be reported to the Principal Investigator, unit management, and the
Ethics Committee after evaluation, followed by completion of an AE
reporting form. All adverse events will be followed until the condition
is stable (discontinuation of observation is considered medically
acceptable) or complete resolution has been achieved.
2.9 Randomization and data collection
Patients successfully enrolled in the study will be randomly assigned
to either the treatment or control group at a 1:1 ratio. The
randomization sequence number will be sealed in an opaque envelope and
provided only to clinicians so that the participants can be assigned to
the treatment regimen accordingly. Since this trial is an open-label
study, participants and physicians will be aware of the assignment
protocol, whereas data analysts and statisticians will not be aware of
the distribution results.
In this study, all participants’ data will be collected, recorded, and
administered in the CRF provided by the Ethics Committee of Shenzhen
Hospital of Guangzhou University of Chinese Medicine (Futian). Data
from the original CRF and biological samples will be stored for an
additional 5 years after the trial ends.
2.10 Sample size calculation
In a previous study ([118]Xu, 2017), JTTF reduced HbA1c levels in
diabetic patients with heat stagnation in liver and stomach after 12
weeks of treatment (treatment group: 6.94±0.88%, control group:
7.64±0.72%, mean difference between the two groups: 0.7%). Considering
a type I error of 5% (α = 0.05) and a power of 85%, the experimental
and control groups were allocated at a ratio of 1:1. The sample size
was calculated using Power Analysis and Sample Size (PASS) software
(version 15.0); a total of 76 patients will be required. According to
the dropout rate of 20%, we set the final sample size to 96, with 48
patients in each group, considering the number of dropout samples.
2.11 Recruitment
Participants in this clinical trial will be recruited mainly from
outpatients and inpatients of the Endocrinology Department of the
Guangzhou University of Chinese Medicine Shenzhen Hospital (Futian).
Patients will also be recruited through posters and publicity on social
media platforms. All examinations during the trial will be free of
charge to participants. Members of the project team will conduct a
detailed protocol explanation and screening of patients who volunteer
to participate in the trial to ensure that participants fully
understand the trial process and the inclusion criteria.
3 Statistical methods
3.1 Data analysis
Data will be analyzed using SPSS software (Version 24.0; IBM Corp.
Armonk, NY, United States). Continuous variables will be represented as
means, standard deviations, medians, and minimum/maximum values. If the
data confirms normal distribution, an independent sample t-test will be
used to assess the differences between the two groups, and a paired
sample t-test to compare the differences between groups before and
after the intervention. Wilcoxon signed-rank test will be used for data
that does not conform to normal distribution. Grade data will be
analyzed using Wilcoxon’s signed-rank test or the CMHχ2 test.
Statistical significance will be set at p < 0.05.
3.2 Monitoring
To maintain high-quality data and ensure the smooth running of the
trial, all protocol implementers and investigators will undergo
systematic and rigorous training. Patient screening, data entry,
dosing, adverse event reporting, dosing records, and biospecimen
extraction will be performed. The data of numbered participants will be
recorded in detail on standardized CRF forms and biospecimens will be
stored under the same number. Data will be recorded independently by
two research assistants, updated in real time, and synchronized on the
CRF form after each visit. Quality control and data review will be
performed regularly by the study monitoring committee to ensure
accuracy and completeness of data entry. The quality and progress of
the project will be evaluated. An interim analysis of the primary
outcome will be conducted to monitor efficacy and safety when 50%
enrollment is reached. This is independent of the sponsor. All patient
information and clinical data will be kept confidential on a
professional data platform.
4 Discussion
NAFLD has a high prevalence worldwide, affecting approximately 25% of
the population ([119]Younossi et al., 2021). It is extremely harmful,
especially when combined with diabetes, and significantly increases the
risk of developing cirrhosis and liver cancer as well as the mortality
rate ([120]Golabi et al., 2018; [121]Younossi and Henry, 2019).
However, there are still no safe and effective drugs for the treatment
of T2DM in combination with NAFLD ([122]Ferguson and Finck, 2021). The
current first-line interventions for both NAFLD, and T2DM with NAFLD
remains lifestyle modification and weight loss, which also has the
disadvantages of low compliance and difficulty in long-term
implementation. Therefore, there is a need for a protocol that
systematically treats T2DM combined with NAFLD as a comorbid disease,
while improving clinical symptoms and combining metabolomics to
identify targeted pathways of action to regulate glucose and lipid
metabolic abnormalities.
This study is a double-armed, parallel-designed, randomized, controlled
clinical trial that aimed to verify the efficacy and safety of JTTF for
glucose-lowering and lipid-modulation and to investigate the
differences in serum and urine metabolites before and after treatment.
The proposed protocol combines clinical evaluation with metabolomics in
an attempt to determine its mechanism of action and pathway of action,
offering the possibility to systematically elucidate the effectiveness
and mechanisms of action of JTTF. Considering the limitations of liver
biopsy implementation, this study focused on subjects with fatty liver
diagnosed by ultrasound to observe changes in visceral fat and
subcutaneous fat area. In addition, patients’ blood lipids, liver
function, BMI, HbA1c level, blood glucose, TCM symptom scores, and
other indicators will be assessed, and the degree of fibrosis and
insulin resistance will be evaluated. Disease onset and progression
will be comprehensively evaluated in this clinical study.
This study has some limitations. First, coronavirus outbreaks remain a
serious concern worldwide and may affect patient follow-up. In
addition, this is a single-center study conducted in China; therefore,
the generalizability of the findings remains unclear. Finally, the
clinical application of JTTF should be expanded to provide evidence for
further mechanistic studies.
5 Trial status
This clinical trial has been registered in China Clinical Trial
Registry (ChiCTR 2100051174) ([123]https://www.chictr.org.cn/index.aspx
). At the time of this manuscript submitted, the state of the trial was
at the recruitment stage. Recruitment is expected to be completed by
October 2022.
Ethics statement
The studies involving human participants were reviewed and approved by
Ethics Committee of Shenzhen Hospital of Guangzhou University of
Chinese Medicine (Futian). The approval number is GZYLL(KY)-2021–025.
The patients/participants provided their written informed consent to
participate in this study.
Author contributions
JX and CP contributed equally to the manuscript and are the co-first
author. YQ drafted the figures and the additional files. TL, YP
provided statistical expertise in clinical trial design. QL would
perform the visceral fat measurement. XZ, PL, XW would recruit the
participants, YF, BC participated in the design of the CRF. JY
critically reviewed this manuscript. All authors approved the final
submitted version of this protocol.
Funding
This study was supported by the National Natural Science Foundation of
China (grant number:81973813), Scientific research project of
Traditional Chinese Medicine Bureau of Guangdong Province (grant
number:20221329) and Shenzhen Science and Technology Innovation Program
(grant number: JCY20190809110015528). The funder was not involved in
the design of this study, its implementation, the writing of this
paper, or its publication.
Conflict of interest
The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or claim
that may be made by its manufacturer, is not guaranteed or endorsed by
the publisher.
Supplementary material
The Supplementary Material for this article can be found online at:
[124]https://www.frontiersin.org/articles/10.3389/fphar.2022.924021/ful
l#supplementary-material
[125]Click here for additional data file.^ (812.2KB, JPEG)
[126]Click here for additional data file.^ (124KB, doc)
[127]Click here for additional data file.^ (17.4KB, docx)
References